Bio-Rad定量PCR说明书.ppt

Bio-rad实时荧光定量PCR仪,BIO-RADGeneExpressionDivision,Outline,RealTimePCRandConventionalPCRIntroductiontoQuantitativePCRCtvalueandReal-TimeQuantitativePCRTheoryLaboratoryTechniquePrimerDesignforReal-TimePCR,RealTimePCRandConventionalPCR,技术背景,本来，PCR技术是为了将样本中微量的DNA模版放大以便研究模版特性随着研究的深入，需要了解样本中基因的表达模式与疾病的关系，这就要了解标本中的DNA原始拷贝数。,DenaturationPrimerAnnealingElongation,Repeat,常规PCRProcess,Intheory,productaccumulationisproportionalto2n,wherenisthenumberofamplificationcyclerepeats,AlinearincreasefollowsexponentialEventuallyplateaus,Cycle#,Realityvs.Theory,Amplificationisexponential,buttheexponentialincreaseislimited:,常规PCR方法的局限性分析：,无法对起始模板准确定量,只能对终产物进行分析对终产物分析，受PCR过程平台效应的干扰，定量准确度难以提高（相对误差1000%）；存在扩增产物的污染问题。必须在扩增后用电泳方法分析,费时费力而且EB有毒无法对扩增反应实时检测,C(t)sfrom96replicatesarenearlyidentical,butthefinalfluorescencevaries.,Cycle,EndPoint,PCR:Theoryvs.Reality,实时定量PCR技术，是指在PCR反应体系中加入荧光基团，利用荧光信号积累实时监测整个PCR进程，使每一个循环变得“可见”，最后通过Ct值和标准曲线对样品中的未知样品的起始浓度进行定量的方法。,Real-TimePCR,QuantitativePCR或RealtimePCR是确定样品中DNA(或cDNA)拷贝数最敏感、最准确的方法,QuantitativePCR/RealtimePCR,C(t)sfrom96replicatesarenearlyidentical,butthefinalfluorescencevaries.,Cycle,EndPoint,C(t),PCR:TheoryvsReality,Quantitativeinformationcomesfrommonitoringtheearlystagesofamplification.,Cycle#,Theoretical,RealLife,LogTargetDNA,Detector,定量的最佳时期,常用的定量方法,相对定量：相对于另一参照样本的量；绝对定量：用标准品作标准曲线来推算未知的样本的量。,QuantitativePCR和常规PCR技术的区别,常规PCR是通过电泳对扩增反应的最终产物进行定性分析（定量不准确）；QuantitativePCR是在PCR反应体系中加入荧光基团，利用荧光信号积累实时监测整个PCR进程，使每一个循环变得“可见”，通过Ct值和标准曲线对样品中的DNA(orcDNA)的起始浓度进行定量的方法（准确定量）。,IntroductiontoQuantitativePCR,PCR仪+监测、分析系统+荧光标记,采用半导体加热和制冷加热速度3.3C/秒，制冷速度2C/秒升降温速度可调控温准确性：0.3,定量PCR仪应该具备的特点：杰出的温度控制,温度梯度选择可以允许使用者在同一次扩增中优化复性温度。,定量PCR仪应该具备的特点：梯度PCR功能,采用多点温控和传感技术，可以实现梯度PCR功能,采用0.2ml的离心管,排管和96孔PCR反应板。降低消耗品成本。,定量PCR仪应该具备的特点：消耗品成本低，样品容量大,同时扩增和检测96个样品,IntroductiontoQuantitativePCR,PCR仪+监测、分析系统+荧光标记,定量PCR仪应该具备的特点：先进的光路设计-多孔样品同时检测,FluorescenceDetection,Multiplexing-同时检测5色荧光,Cycle,LogRelativeFluorescence,Multiplexing,定量PCR仪应该具备的特点：开放性的系统,可兼容的定量方法：SYBRGreenI、Taqman探针、Beacon探针、FRET探针,可兼容的突变检测方法：熔点曲线功能、MGB探针,可兼容的试剂种类：所有国产与进口试剂,iQ5DataAnalysisSoftware,iQ5DataAnalysisSoftware,定量PCR仪应该具备的特点：分析软件功能强大简洁直观,异常数据或情况提示,标准曲线定量计算,自动数据分析,多种报告单模式可供选择-尤其重要,基因表达分析,IntroductiontoQuantitativePCR,PCR仪+监测、分析系统+荧光标记,DetectionChemistries,Non-specificDNAbindingdyesSYBRGreenISYBRGoldEthidiumBromideSpecificHybridizationProbesCleavageProbes(TaqManTM)MolecularbeaconsScorpionsTMAmplifluorTMLUXTMdual-oligoFRETpairs,QuantitativePCRDetectionChemistries,DNABindingDyes,5,Extension,ExtensionContinuedApplyExcitationWavelength,Repeat,DNAbindingdyes,DNABindingDyes,Advantages!InexpensivecomparedtohybridizationprobesNoadditionaldesignworkthantheprimerusedforPCRreactionHoweverNottemplatespecific,willbindALLdoublestrandedDNAinducingprimer-dimerandunspecificampliconformationMultiplexassaysnotpossible,SYBRGreenIExperiment,Typical“firststep”experiment:EvaluatePrimerSpecificityUsingMeltCurveAnalysisEvaluatePrimerPairEfficienciesByrunningserialdilutionsoftemplateasstandardsIdentifySub-OptimalaspectsofassayOptimizefurtherwiththermalgradient,etc.,CleavageProbes(TaqManTM),5,3,Q,F,Taq,5,q,3,l,3,5,杂交探针的互补区在引物间5端连有一个荧光reporter,通常是荧光素3端连有一个Quencher,通常是TAMRA荧光素被488nm光激发，发射光为520nm520nm光能激发TAMRA，发射光为570nm,TaqMan,Cleavage-basedassay:TaqManTM,d.NTPs,ThermalStableDNAPolymerase,Primers,AddMasterMixandSample,Denaturation,Annealing,ReactionTube,l,5,3,ExtensionStep,1.StrandDisplacement,2.Cleavage,3.PolymerizationComplete,4.Detection,l,Cleavage-basedassay:TaqManTM,CleavageProbes(TaqManTM),Advantages!GeneratesarobustcumulativefluorescencesignalSimpletodesignandsynthesizecomparedtootherhybridizationprobes(i.e.beacons)IdealapproachformultiplexassaysSNP(SingleNucleotidePolymorphism)assaypossibleHoweverMoreexpensivethanDNAbindingdyes,MolecularBeacons,R,Q,MolecularBeacon,5,3,R,Q,探针有核心的杂交区探针有自互补末端在未杂交时探针呈发夹状报道子,荧光素在探针的5端猝灭子在探针的3端,分子Beacons,当探针是发夹结构时，在报道子和猝灭子间有直接的能量转移,Beacon构造,MolecularBeacons,Advantages!GoodforSNP(SingleNucleotidePolymorphism)detectionMultiplexassayspossibleHoweverMolecularBeaconsAreDIFFICULTtoDesignandSynthesizeDoesNOTgenerateacumulativefluorescencesignal,muchweakersignalthanthe5NucleaseAssay(Cleavageprobe)Expensive!,AFewWordsaboutProbes,HybridizationoligosshouldhaveTms612degreeshigherthantheassociatedprimers.Avoidsecondarystructureinthecomplementaryregionoftheprobe.AvoidGsatthe5endoftheprobesequence.Useoligoanalysistoolstocheckprobefor:DimerizationSecondaryStructureCrossReactivitywithPrimers,EachmethodhasadvantagesanddisadvantagesBio-RadReal-TimeInstrumentationisequippedtohandleallchemistriesOnemethodmaybemoreappropriateforanapplicationoveranother,WhichMethodtoUse?,Outline,RealTimePCRandConventionalPCRIntroductiontoQuantitativePCRCtvalueandReal-TimeQuantitativePCRTheoryLaboratoryTechniquePrimerDesignforReal-TimePCR,CtvalueandReal-TimeQuantitativePCRTheory,基线（baseline)阈值(threshold),基线（baseline）的设置以PCR反应的前15个循环的荧光信号作为荧光本底信号阈值(threshold)的设置一般是3-15个循环的荧光信号的标准偏差的10倍。PCR扩增信号进入相对稳定对数增长期时的荧光值。,阈值循环数Ct,PCR扩增过程中荧光信号开始由本底进入指数增长阶段所对应的循环次数，也就是荧光信号达到阈值时的循环次数。,从图中的重复实验中可以直观地看到，随着PCR反应过程，荧光信号从基线经一个转折点进入指数期、线性期和最终的平台期，尽管平台期DNA拷贝数波动很大，CT值却是相对固定的。,Ct值与起始模板的关系,ThresholdCycle,CT,如果用不同浓度模版DNA作PCR，可以看出模版浓度越高，CT值越小。,模板起始浓度越高，Ct值越小Ct值与模板起始拷贝数的对数存在线性关系,Ct值与模板起始浓度的关系,如果模板浓度增加1倍，Ct值就提前1个循环到达如果模板浓度减少1倍，Ct值就滞后1个循环到达,1CTDifference=2folddifferenceinstartingtemplateamount3.3CTDifference=10folddifferenceinstartingtemplateamount,ProductT=(Template0)2nWheren=NumberofCycles,MathematicImplications,IdealPCR,ThresholdCycle,Ct,isareliableindicatorofinitialcopynumber,利用已知起始拷贝数的标准品可作出标准曲线，其中横坐标代表起始拷贝数的对数，纵坐标代表Ct值。因此，只要获得未知样品的Ct值，即可从标准曲线上计算出该样品的起始拷贝数。,AbsoluteandRelativequantification,Quantification,NormalizationofRT-PCRusingreferencegenes,DetermineschangesinsteadystatetranscriptionofageneorgenesExpressionofthegene/sofinterestisnormalizedagainstareferencegene/s(housekeepinggene/s)withnoorinsignificantexpressionvariationExamplesofsomereferencegenes/housekeepinggenesused:b-Actin,GAPDH,18srRNAb-2Microglobulin,Cyclophilins,Beta-ActinOrnithinedecarboxylase(ODC)S-adenysylmethioninedecarboxylase(SAMDC)HumanProstateandThymus,Real-TimeMultiplexPCR:GeneExpression,Real-timeMultiplexGeneExpression,Beta-Actin,ODC,SAMDC,ProstateCt:23.25ThymusCt:26.93,ProstateCt:23.10ThymusCt:25.53,ProstateCt:21.25ThymusCt:21.90,OnlypossiblewithDNABindingdyes(SYBRGreenI)andaftercompletedrealtimePCRDeterminesthetemperatureatwhich50%oftheDNAmoleculesseparateintotwostrands-or“melts”apartDiscriminatesbyMeltingTemperature(Tm),Tmisdependenton:-sequence(G/Ccontent)-length,WhatisMeltCurveAnalysis?,Fluorescencevs.Temperature,Meltcurveshowingtwoamplifiedproducts,MeltCurveCheckspecificityofthereaction,CollectingatHigherTemperatures;doesthatworktoavoiddetectionofprimerdimers?,Collectingat82Cwouldrecordspecificproductonly,PrimerDimersImpactontheAssay,Outline,RealTimePCRandConventionalPCRIntroductiontoQuantitativePCRCtvalueandReal-TimeQuantitativePCRTheoryLaboratoryTechniquePrimerDesignforReal-TimePCR,LaboratoryTechnique,GoodLaboratoryTechnique,Donotunderestimatetheimportanceofusing:ScrewcaptubesAerosolbarriertipsHot-startpolymeraseMastermixesReplicatesSerialdilutionsAndthegoldenrule:Pipetonlyonceintoeachw